Polypeptides Having Protease Activity

ABSTRACT

The present invention relates to isolated polypeptides having protease activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides in e.g. animal feed and detergents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/237,189filed on Feb. 5, 2014, now pending, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2012/066099 filed Aug. 17, 2012, which claimspriority or the benefit under 35 U.S.C. 119 of U.S. provisionalapplication No. 61/525,906 filed Aug. 22, 2011. The content of eachapplication is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having proteaseactivity and isolated nucleic acid sequences encoding the proteases. Theinvention also relates to nucleic acid constructs, vectors, and hostcells, including plant and animal cells, comprising the nucleic acidsequences, as well as methods for producing and using the proteases, inparticular the use of the proteases in animal feed, and detergents.

2. Background of the Invention

In the use of proteases in animal feed (in vivo), and/or the use of suchproteases for treating vegetable proteins (in vitro) it is noted thatproteins are essential nutritional factors for animals and humans. Mostlivestock and human beings get the necessary proteins from vegetableprotein sources. Important vegetable protein sources are e.g. oilseedcrops, legumes and cereals.

When e.g. soybean meal is included in the feed of mono-gastric animalssuch as pigs and poultry, a significant proportion of the soybean mealsolids is not digested efficiently (the apparent ileal proteindigestibility in piglets, growing pigs and poultry such as broilers,laying hens and roosters is only around 80%).

The gastrointestinal tract of animals consists of a series of segmentseach representing different pH environments. In mono-gastric animalssuch as pigs and poultry and many fish the stomach exhibits stronglyacidic pH as low as pH 1-2, while the intestine exhibit a more neutralpH in the area pH 6-7. Poultry in addition to stomach and intestine alsohave a crop preceding the stomach, pH in the crop is mostly determinedby the feed ingested and hence typically lies in the range pH 4-6.Protein digestion by a protease may occur along the entire digestivetract, given that the protease is active and survives the conditions inthe digestive tract. Hence, proteases which are highly acid stable forsurvival in the gastric environment and at the same time are efficientlyactive at broad physiological pH of the target animal are especiallydesirable.

Also, animal feed is often formulated in pelleted form, where steam isapplied in the pelleting process. It is therefore also desireable thatproteases used in animal feed are capable to remain active afterexposure to steam treatment

Polypeptides Having Protease Activity

Polypeptides having protease activity, or proteases, are sometimes alsodesignated peptidases, proteinases, peptide hydrolases, or proteolyticenzymes. Proteases may be of the exo-type that hydrolyse peptidesstarting at either end thereof, or of the endo-type that act internallyin polypeptide chains (endopeptidases). Endopeptidases show activity onN- and C-terminally blocked peptide substrates that are relevant for thespecificity of the protease in question.

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. This definition of protease also applies to theprotease-part of the terms “parent protease” and “protease variant,” asused herein. The term “protease” includes any enzyme belonging to the EC3.4 enzyme group (including each of the thirteen subclasses thereof).The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, AcademicPress, San Diego, Calif., including supplements 1-5 published in Eur. J.Biochem. 223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J.Biochem. 237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J.Biochem. 264: 610-650 (1999); respectively. The nomenclature isregularly supplemented and updated; see, e.g.,www.chem.qmw.ac.uk/iubmb/enzyme/index.html.

The proteases of the invention and for use according to the inventionare selected from the group consisting of:

(a) proteases belonging to the EC 3.4.21. enzyme group; and/or

(b) Serine proteases of the peptidase family S1, or more specificallyS1A; as described in Biochem. J. 290: 205-218 (1993) and in MEROPSprotease database, release, 9.4 (31 Jan. 2011) (www.merops.ac.uk). Thedatabase is described in Rawlings et al., 2010, MEROPS: the peptidasedatabase. Nucleic Acids Res. 38: D227-D233.

More specifically the proteases of the invention are those that prefer ahydrophobic aromatic aa residue in the P1 position.

For determining whether a given protease is a Serine protease, and afamily S1A protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

The peptidases of family S1 contain the catalytic triad His, Asp and Serin that order. Mutation of any of the amino acids of the catalytic triadwill result in loss of enzyme activity. The amino acids of the catalytictriad of the S1 protease 1 from Kribbella solani (SEQ ID NO: 2) andKribbella aluminosa (SEQ ID NO: 4) are probably positions His-138,Asp-168 and Ser-250.

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperaturesare 5, 10, 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90,or 95° C. Examples of protease substrates are casein, such asAzurine-Crosslinked Casein (AZCL-casein), or suc-AAPF-pNA. Examples ofsuitable protease assays are described in the experimental part.

DESCRIPTION OF THE RELATED ART

Proteases isolated from Kribbella, and Streptomyces are known in theart. A protease from Kribbella flavida is disclosed in Lucas, S. et al.“The complete genome of Kribbella flavida DSM 17836”; Submitted(September-2009) to the EMBL/GenBank/DDBJ databases (SWISSPROT: C1WJ16;SEQ ID NO: 6). The sequence has 80.23% identity to the sequence of SEQID NO: 2 and 80.81% identity to the sequence of SEQ ID NO: 4 for themature protease. The DNA sequence of the reference (SEQ ID NO: 5) has anidentity of 81.6% to the sequence of sequence of SEQ ID NO: 1 and 85.51%identity to the sequence of SEQ ID NO: 3.

A protease, Streptogrisin B, is disclosed in Henderson et al., 1987,“Characterization and structure of genes for proteases A and B fromStreptomyces griseus”, J. Bacteriol. 169: 3778-3784. Sequence identitiesfor this protease are lower than those indicated above:

The present invention provides polypeptides having protease activity andpolynucleotides encoding the polypeptides. The proteases of theinvention are serine proteases of the peptidase family S1A. Theproteases of the invention exhibit surprising pH properties, especiallypH stability and pH-activity properties which makes them interestingcandidates for use in animal feed. The proteases of the invention thusare active on Suc-Ala-Ala-Pro-Phe-pNA within a broad range from pH 4-11and exhibit especially high activity in the range pH 6-11, are active ona feed relevant soybean meal-maize meal substrate within a broadphysiological pH range from pH 3-7 and retains more than 80% activityafter being subjected for 2 hours to pH as low as 2.

The use of proteases in animal feed to improve digestion of proteins inthe feed is known. WO 95/28850 discloses the combination of a phytaseand one or more microbial proteolytic enzymes to improve the solubilityof vegetable proteins. WO 01/58275 discloses the use of acid stableproteases of the subtilisin family in animal feed. WO 01/58276 disclosesthe use in animal feed of acid-stable proteases related to the proteasederived from Nocardiopsis sp. NRRL 18262 (the 10R protease), as well asa protease derived from Nocardiopsis alba DSM 14010. WO 2004/072221, WO2004/111220, WO 2004/111223, WO 2005/035747, and WO 2005/123911 discloseproteases related to the 10R protease and their use in animal feed.Also, WO 04/072279 discloses the use of other proteases.

WO 2004/034776 discloses the use of a subtilisin/keratinase, PWD-1 fromB. licheniformis in the feed of poultry. WO 2004/077960 discloses amethod of increasing digestibility of forage or grain in ruminants byapplying a bacterial or fungal protease.

Commercial products comprising a protease and marketed for use in animalfeed include RONOZYME® ProAct (DSM NP/Novozymes), Axtra® (Danisco),Avizyme® (Danisco), Porzyme® (Danisco), Allzyme™ (Alltech), Versazyme®(BioResources, Int.), Poultrygrow™ (Jefo) and Cibenza® DP100 (Novus).

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having proteaseactivity selected from the group consisting of:

(a) a polypeptide having at least 85% sequence identity to the maturepolypeptide of either SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide having at least 86%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3;

(c) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2; or SEQ ID NO: 4; and

(d) a fragment of a polypeptide of (a), (b), or (c), that has proteaseactivity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention, nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides, and to methods of producing the polypeptides.

The present invention also relates to methods for preparing acomposition for use in animal feed, for improving the nutritional valueof an animal feed, and methods of treating proteins to be used in animalfeed compositions.

Furthermore the present invention also relates to the use of theproteases in detergent compositions and such detergent compositions.

OVERVIEW OF SEQUENCE LISTING

SEQ ID NO: 1 is the DNA sequence as isolated from the Kribbella solani.

SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1

SEQ ID NO: 3 is the DNA sequence as isolated from the Kribbellaaluminosa

SEQ ID NO: 4 is the amino acid sequence as deduced from SEQ ID NO: 3

SEQ ID NO: 5 is the DNA sequence from Kribbella flavida (EMBL:CP001736)

SEQ ID NO: 6 is the amino acid sequence from Kribbella flavida (Lucas etal., “The complete genome of Kribbella flavida DSM 17836.”UNIPROT:D2Q1F6)

SEQ ID NO: 7 is the DNA sequence of the 10R protease (WO 05/035747, SEQID NO: 1)

SEQ ID NO:8 is the amino acid sequence of the 10R protease (WO2005/035747, SEQ ID NO: 2)

SEQ ID NO: 9 is the Kribbella solani S1 peptidase specific primerforward.

SEQ ID NO: 10 is the Kribbella solani S1 peptidase specific primerreverse.

SEQ ID NO: 11 is the Kribbella aluminosa S1 peptidase specific primerforward.

SEQ ID NO: 12 is the Kribbella aluminosa S1 peptidase specific primerreverse.

SEQ ID NO: 13 Upstream flanking fragment specific primer forward.

SEQ ID NO: 14 Upstream flanking fragment specific primer reverse.

SEQ ID NO: 15 Downstream flanking fragment specific primer forward.

SEQ ID NO: 16 Downstream flanking fragment specific primer reverse.

SEQ ID NO: 17 is a Bacillus lentus secretion signal.

Identity Matrix of Sequences

SEQ ID SEQ ID NO: 6 NO: 8 SEQ ID SEQ ID Kribbella 10R Protein NO: 2 NO:4 flavida protease SEQ ID NO: 2 100 95 80.23 46.41 SEQ ID NO: 4 10080.81 47.51 SEQ ID NO: 6 100 60.23 SEQ ID NO: 8 100 SEQ ID SEQ ID NO: 5NO: 7 SEQ ID SEQ ID Kribbella 10R DNA NO: 1 NO: 3 flavida protease SEQID NO: 1 100 90.76 81.82 70.96 SEQ ID NO: 3 90.76 100 85.51 72.92 SEQ IDNO: 5 100 72.68 SEQ ID NO: 7 100

DEFINITIONS

Protease activity: The term “protease activity” means a proteolyticactivity (EC 3.4). Proteases of the invention are endopeptidases (EC3.4.21). There are several protease activity types: The three mainactivity types are: trypsin-like where there is cleavage of amidesubstrates following Arg or Lys at P1, chymotrypsin-like where cleavageoccurs following one of the hydrophobic amino acids at P1, andelastase-like with cleavage following an Ala at P1. For purposes of thepresent invention, protease activity is determined according to theprocedure described in “Materials and Methods” below.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the protease activity ofthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

An “isolated polypeptide” is at least 1% pure, e.g., at least 5% pure,at least 10% pure, at least 20% pure, at least 40% pure, at least 60%pure, at least 80% pure, and at least 90% pure, as determined bySDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well known recombinantmethods or by classical purification methods.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 188 in the numbering of SEQ ID NO: 2,amino acids −105 to −75 in the numbering of SEQ ID NO: 2 is a signalpeptide. In a further aspect, the mature polypeptide is amino acids 1 to189 in the numbering of SEQ ID NO: 4, amino acids −105 to −75 in thenumbering of SEQ ID NO: 4 is a signal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving protease activity. In one aspect, the mature polypeptide codingsequence is nucleotides 316 to 879 in the numbering of SEQ ID NO: 1.Further nucleotides 1 to 90 in the numbering of SEQ ID NO: 1 encode asignal peptide. In a further aspect, the mature polypeptide codingsequence is nucleotides 316 to 882 in the numbering of SEQ ID NO: 1.Further nucleotides 1 to 90 in the numbering of SEQ ID NO: 1 encode asignal peptide.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 5.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has protease activity. In oneaspect, a fragment contains at least 168 amino acid residues (e.g.,amino acids 11 to 178 of SEQ ID NO: 2), at least 178 amino acid residues(e.g., amino acids 6 to 183 of SEQ ID NO: 2); or correspondingly for SEQID NO: 4 a fragment contains at least 169 amino acid residues (e.g.,amino acids 11 to 179 of SEQ ID NO: 4) or at least 180 amino acidresidues (e.g., amino acids 5 to 184 of SEQ ID NO: 4).

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving protease activity. In one aspect, a subsequence contains at least504 nucleotides (e.g., nucleotides 346 to 849 of SEQ ID NO: 1), or e.g.,at least 534 nucleotides (e.g., nucleotides 331 to 864 of SEQ ID NO: 1);or correspondingly for SEQ ID NO: 3 a fragment contains at least 507nucleotides (e.g. nucleotides 346 to 852 of SEQ ID NO: 3) or e.g. atleast 540 nucleotides (e.g. nucleotides 328 to 867 of SEQ ID NO: 3).

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” means apolynucleotide that is modified by the hand of man relative to thatpolynucleotide as found in nature. In one aspect, the isolatedpolynucleotide is at least 1% pure, e.g., at least 5% pure, more atleast 10% pure, at least 20% pure, at least 40% pure, at least 60% pure,at least 80% pure, at least 90% pure, and at least 95% pure, asdetermined by agarose electrophoresis. The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” means a polynucleotide preparation free of otherextraneous or unwanted nucleotides and in a form suitable for use withingenetically engineered polypeptide production systems. Thus, asubstantially pure polynucleotide contains at most 10%, at most 8%, atmost 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, andat most 0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. Preferably, thepolynucleotide is at least 90% pure, e.g., at least 92% pure, at least94% pure, at least 95% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99% pure, and at least 99.5% pure by weight.The polynucleotides of the present invention are preferably in asubstantially pure form.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptideof the present invention. Each control sequence may be native or foreignto the polynucleotide encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Variant: The term “variant” means a polypeptide having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion of one or more (several) amino acid residues at one or more(several) positions. A substitution means a replacement of an amino acidoccupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding 1-3 amino acids adjacent to an amino acid occupying a position.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having ProteaseActivity

The present invention relates to isolated polypeptides having proteaseactivity selected from the group consisting of:

(a) a polypeptide having at least 85% sequence identity to the maturepolypeptide of SEQ ID NO: 2 and SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions, or very high stringency conditions with

-   -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,        and/or    -   (ii) the mature polypeptide coding sequence of SEQ ID NO: 3, or    -   (iii) the full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 86%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3; and/or (d) a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2; and SEQ ID NO: 4.

The present invention relates to isolated polypeptides having a sequenceidentity to the mature polypeptide of SEQ ID NO: 2 of at least 85%,e.g., at least 87%, at least 89%, at least 90%, at least 93%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have protease activity. In one aspect, the polypeptides differ byno more than ten amino acids, e.g., by nine amino acids, by eight aminoacids, by seven amino acids, by six amino acids, by five amino acids, byfour amino acids, by three amino acids, by two amino acids, and by oneamino acid from the mature polypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 87% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 89% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 90% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 93% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 95% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 96% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 97% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 98% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 99% sequence identity to thepolypeptide of SEQ ID NO: 2.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 100% sequence identity to thepolypeptide of SEQ ID NO: 2.

The present invention relates to isolated polypeptides having a sequenceidentity to the mature polypeptide of SEQ ID NO: 4 of at least 85%,e.g., at least 87%, at least 89%, at least 90%, at least 93%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%,which have protease activity. In one aspect, the polypeptides differ byno more than twentyfive amino acids, e.g., by twenty amino acids, byfifteen amino acids, by ten amino acids, by nine amino acids, by eightamino acids, by seven amino acids, by six amino acids, by five aminoacids, by four amino acids, by three amino acids, by two amino acids,and by one amino acid from the mature polypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 87% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 89% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 90% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 93% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 95% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 96% sequence identity to thepolypeptide of SEQ ID NO: 4. An embodiment of the invention is apolypeptide or a polypeptide encoded by a polynucleotide having at least97% sequence identity to the polypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 98% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 99% sequence identity to thepolypeptide of SEQ ID NO: 4.

An embodiment of the invention is a polypeptide or a polypeptide encodedby a polynucleotide having at least 100% sequence identity to thepolypeptide of SEQ ID NO: 4.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or an allelicvariant thereof; or is a fragment thereof having protease activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. In another preferredaspect, the polypeptide comprises or consists of amino acids 1 to 188 ofSEQ ID NO: 2, or amino acids 1 to 189 of SEQ ID NO: 4.

The present invention also relates to isolated polypeptides havingprotease activity that are encoded by polynucleotides that hybridizeunder high stringency conditions, or very high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQID NO: 3, (ii) [the genomic DNA sequence comprising] the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii)the full-length complementary strand of (i) or (ii) (J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3; or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO: 2 or SEQ IDNO: 4, or a fragment thereof, may be used to design nucleic acid probesto identify and clone DNA encoding polypeptides having protease activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having protease activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that is homologous with SEQ ID NO: 1 or SEQ ID NO: 3; ora subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:3; [the genomic DNA sequence comprising] the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3; its full-length complementarystrand; or a subsequence thereof; under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3. In another aspect, the nucleicacid probe is a fragment thereof. In another aspect, the nucleic acidprobe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2or SEQ ID NO: 4 or a fragment thereof. In another preferred aspect, thenucleic acid probe is SEQ ID NO: 1 or SEQ ID NO: 3.

For long probes of at least 100 nucleotides in length, high to very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and either 25% formamide for very low and lowstringencies, 35% formamide for medium and medium-high stringencies, or50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 65° C. (high stringency), and at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

The present invention also relates to isolated polypeptides havingprotease activity encoded by polynucleotides having a sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1 of at least86%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 86%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 90%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 95%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 96%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 97%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 98%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 99%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of at least 100%.

The present invention also relates to isolated polypeptides havingprotease activity encoded by polynucleotides having a sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 3 of at least86%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 86%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 90%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 95%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 96%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 97%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 98%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 99%.

An embodiment of the invention is polypeptides having protease activityencoded by polynucleotides having a sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of at least 100%.

In particular embodiments, the parent proteases and/or the proteasevariants of the invention and for use according to the invention areselected from the group consisting of:

(a) Proteases belonging to the EC 3.4.21 enzyme group; and

(b) Serine proteases of peptidase family S1A; as described in Biochem.J.290:205-218 (1993) and in MEROPS protease database, release 9.5(www.merops.ac.uk). The database is described in Rawlings et al., 2010,MEROPS: the peptidase database, Nucleic Acids Res. 38: D227-D233.

For determining whether a given protease is a Serine protease, and afamily S1A protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

The present invention also relates to variants comprising asubstitution, deletion, and/or insertion of one or more (or several)amino acids of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4,or a homologous sequence thereof. Preferably, amino acid changes are ofa minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like. Essential amino acids in a parent polypeptide can beidentified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, 1989, Science 244: 1081-1085). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for proteaseactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to the parentpolypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 isnot more than 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20.

The polypeptide may be hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The polypeptide may be a fused polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusedpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The proteases of the invention exhibit surprising pH properties,especially pH stability and pH-activity properties, especially at low pHvalues, which makes them interesting candidates for use in animal feedand detergents.

EMBODIMENTS

In certain embodiments of the invention, the protease of the inventionexhibits beneficial thermal properties such as thermostability, steamstability, etc and/or pH properties, such as acid stability, pH optimum,etc.

An embodiment of the invention is isolated polypeptides having improvedprotease activity between pH 4 and 9, such as between pH 5 and 8, suchas at pH 5, at pH 6, at pH 7 or at pH 8, at 25° C. compared to protease10R.

An additional embodiment of the invention is improved protease activityon soybean-maize meal between pH 3.0 and 6.0 at 40° C., such as at pH3.0, at pH 4.0, at pH 5.0 or at pH 6.0, compared to protease 10R.

Acidity/Alkalinity Properties

In certain embodiments of the invention the protease of the inventionexhibits beneficial properties in respect of pH, such as acid stability,pH optimum, etc. Stability of the protease at a low pH is beneficialsince the protease can have activity in the intestine after passingthrough the stomach. In one embodiment of the invention the proteaseretains >95% activity after 2 hours at pH 3 as determined using themethod described in Example 3.

Thermostability

Thermostability may be determined as described in Example 6, i.e. usingDSC measurements to determine the denaturation temperature, T_(d), ofthe purified protease protein. The Td is indicative of thethermostability of the protein: The higher the T_(d), the higher thethermostability. Accordingly, in a preferred embodiment, the protease ofthe invention has a T_(d) which is higher than the T_(d) of a referenceprotease, wherein T_(d) is determined on purified protease samples(preferably with a purity of at least 90% or 95%, as determined bySDS-PAGE).

In preferred embodiments, the thermal properties such as heat-stability,temperature stability, thermostability, steam stability, and/orpelleting stability as provided by the residual activity, denaturationtemperature T_(d), or other parameter of the protease of the inventionis higher than the corresponding value, such as the residual activity orT_(d), of the protease of SEQ ID NO: 5, more preferably at least 101%thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, orat least 110% thereof. Even more preferably, the value of the parameter,such as residual activity or T_(d), of the protease of the invention isat least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% ofthe value for the protease of SEQ ID NO: 5.

In still further particular embodiments, the thermostable protease ofthe invention has a melting temperature, T_(m) (or a denaturationtemperature, T_(d)), as determined using Differential Scanningcalorimetry (DSC) as described in example 10 (i.e. in 20 mM sodiumacetate, pH 4.0), of at least 50° C. In still further particularembodiments, the T_(m) is at least 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or at least 100° C.

Steam Stability

Steam stability may be determined as described in Example 7 bydetermining the residual activity of protease molecules after steamtreatment at 85° C. or 90° C. for a short time.

Pelleting Stability

Pelleting stability may be determined as described in Example 8 by usingenzyme granulate pre-mixed with feed. From the mixer the feed isconditioned with steam to 95° C. After conditioning the feed is pressedto pellets and the residual activity determined.

Sources of Polypeptides Having Protease Activity

A polypeptide having protease activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a polypeptide having protease activity from agram-positive bacterium within a phylum such Actinobacteria or from agram-negative bacterium within a phylum such as Proteobacteria.

In one aspect, the polypeptide is a protease from a bacterium of theclass Actinobacteria, such as from the order Actinomycetales, or fromthe suborder Propionibacterineae, or from the family Nocardioidaceae, orfrom the genera Kribbella, Saccharomonospora, Saccharopolyspora; orAmycolatopsis.

Strains of these taxa are readily accessible to the public in a numberof culture collections, such as the American Type Culture Collection(ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH(DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) using the above-mentioned probes. Techniques for isolatingmicroorganisms from natural habitats are well known in the art. Thepolynucleotide encoding the polypeptide may then be obtained bysimilarly screening a genomic or cDNA library of another microorganismor mixed DNA sample. Once a polynucleotide encoding a polypeptide hasbeen detected with the probe(s), the polynucleotide can be isolated orcloned by utilizing techniques that are well known to those of ordinaryskill in the art (see, e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ofKribbella, or another or related organism from the Actinomycetales andthus, for example, may be an allelic or species variant of thepolypeptide encoding region of the polynucleotide.

The present invention also relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orSEQ ID NO: 3 of at least 86%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100%, which encode apolypeptide having protease activity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant may be constructed on the basis of the polynucleotidepresented as the mature polypeptide coding sequence of SEQ ID NO: 1 orSEQ ID NO: 3, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not result in a change in the aminoacid sequence of the polypeptide, but which correspond to the codonusage of the host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions that may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) the full-length complementarystrand of (i) or (ii); or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 1or SEQ ID NO: 3, the mature polypeptide coding sequence of SEQ ID NO: 1,or a subsequence of SEQ ID NO: 1 or SEQ ID NO: 3 that encodes a fragmentof SEQ ID NO: 2 or SEQ ID NO: 4 having protease activity, such as thepolynucleotide of nucleotides 316 to 879 of SEQ ID NO: 1 or nucleotides316 to 882 SEQ ID NO: 3.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide of the present invention. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the polypeptide at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a polypeptideof the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Brevibacillus, Clostridium, Geobacillus, Lactobacillus,Lactococcus, Paenibacillus, and Streptomyces. Gram-negative bacteriainclude, but are not limited to E. coli, and Pseudomonas.

The bacterial host cell may be any Bacillales cell including, but notlimited to, Bacillus amyloliquefaciens, Brevibacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lentus,Bacillus licheniformis, Geobacillus stearothermophilus, Bacillussubtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207, by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Kribbella. In a morepreferred aspect, the cell is Kribbella solani or Kribbella aluminosa.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods well known in the art. Forexample, the cell may be cultivated by shake flask cultivation, andsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing a polypeptide is used as asource of the polypeptide.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotide of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing thepolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding a polypeptide into the planthost genome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding apolypeptide can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the present invention encompasses notonly a plant directly regenerated from cells which have been transformedin accordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention, or a portion of a DNA constructprepared in accordance with the present invention. Crossing results inthe introduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to compositions comprising a proteaseof the present invention. Preferably, the compositions are enriched insuch a protease. The term “enriched” indicates that the proteaseactivity of the composition has been increased, e.g., with an enrichmentfactor of at least 1.1.

The composition may comprise a protease of the present invention as themajor enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, bymicroorganisms such as bacteria or fungi or by plants or by animals. Thecompositions may be prepared in accordance with methods known in the artand may be in the form of a liquid or a dry composition. For instance,the composition may be in the form of a granulate or a microgranulate.The protease may be stabilized in accordance with methods known in theart.

Uses

The present invention is also directed to methods for using thepolypeptides having protease activity, or compositions thereof.

Animal Feed

The present invention is also directed to methods for using theproteases having protease activity in animal feed, as well as to feedcompositions and feed additives comprising the proteases of theinvention.

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goats, and cattle, e.g. beef cattle,cows, and young calves. In a particular embodiment, the animal is anon-ruminant animal. Non-ruminant animals include mono-gastric animals,e.g. pigs or swine (including, but not limited to, piglets, growingpigs, and sows); poultry such as turkeys, ducks and chicken (includingbut not limited to broiler chicks, layers); horses (including but notlimited to hotbloods, coldbloods and warm bloods), young calves; andfish (including but not limited to salmon, trout, tilapia, catfish andcarps; and crustaceans (including but not limited to shrimps andprawns).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 12 of WO 01/58275). In other particular embodiments the proteasepreparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95%pure as determined by this method.

A well-defined protease preparation is advantageous. For instance, it ismuch easier to dose correctly to the feed a protease that is essentiallyfree from interfering or contaminating other proteases. The term dosecorrectly refers in particular to the objective of obtaining consistentand constant results, and the capability of optimising dosage based uponthe desired effect.

For the use in animal feed, however, the protease need not be that pure;it may e.g. include other enzymes, in which case it could be termed aprotease preparation.

The protease preparation can be (a) added directly to the feed (or useddirectly in a protein treatment process), or (b) it can be used in theproduction of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original protease preparation, whether used according to(a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods.

Such protease preparation may of course be mixed with other enzymes.

The protein may be an animal protein, such as meat and bone meal,feather meal, and/or fish meal; or it may be a vegetable protein.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g. soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

In a particular embodiment of a treatment process the protease(s) inquestion is affecting (or acting on, or exerting its hydrolyzing ordegrading influence on) the proteins, such as vegetable proteins orprotein sources. To achieve this, the protein or protein source istypically suspended in a solvent, eg an aqueous solvent such as water,and the pH and temperature values are adjusted paying due regard to thecharacteristics of the enzyme in question. For example, the treatmentmay take place at a pH-value at which the activity of the actualprotease is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or atleast 90%. Likewise, for example, the treatment may take place at atemperature at which the activity of the actual protease is at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%. The abovepercentage activity indications are relative to the maximum activities.The enzymatic reaction is continued until the desired result isachieved, following which it may or may not be stopped by inactivatingthe enzyme, e.g., by a heat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning, e.g., that theprotease is added to the proteins, but its hydrolysing influence is soto speak not switched on until later when desired, once suitablehydrolysing conditions are established, or once any enzyme inhibitorsare inactivated, or whatever other means could have been applied topostpone the action of the enzyme.

In one embodiment the treatment is a pre-treatment of animal feed orproteins for use in animal feed, i.e., the proteins are hydrolyzedbefore intake.

The term improving the nutritional value of an animal feed meansimproving the availability of nutrients in the feed. In this inventionimproving the nutritional values refers in particular to improving theavailability of the protein fraction of the feed, thereby leading toincreased protein extraction, higher protein yields, and/or improvedprotein utilization. When the nutritional value of the feed isincreased, the protein and/or amino acid digestibility is increased andthe growth rate and/or weight gain and/or feed conversion (i.e. theweight of ingested feed relative to weight gain) of the animal might beimproved.

The protease can be added to the feed in any form, be it as a relativelypure protease, or in admixture with other components intended foraddition to animal feed, i.e. in the form of animal feed additives, suchas the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives, e.g.premixes.

Apart from the protease of the invention, the animal feed additives ofthe invention contain at least one fat-soluble vitamin, and/or at leastone water soluble vitamin, and/or at least one trace mineral, and/or atleast one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.carotenoids such as beta-carotene, astaxanthin, and lutein; stabilisers;growth improving additives and aroma compounds/flavorings, e.g. creosol,anethol, deca-, undeca- and/or dodeca-lactones, ionones, irone,gingerol, piperidine, propylidene phatalide, butylidene phatalide,capsaicin and/or tannin; antimicrobial peptides; polyunsaturated fattyacids (PUFAs); reactive oxygen generating species; also, a support maybe used that may contain, for example, 40-50% by weight of wood fibres,8-10% by weight of stearine, 4-5% by weight of curcuma powder, 4-58% byweight of rosemary powder, 22-28% by weight of limestone, 1-3% by weightof a gum, such as gum arabic, 5-50% by weight of sugar and/or starch and5-15% by weight of water.

A feed or a feed additive of the invention may also comprise at leastone other enzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase(EC 3.2.1.22); further protease (EC 3.4), phospholipase A1 (EC3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4);amylase such as, for example, alpha-amylase (EC 3.2.1.1); and/orbeta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

In a particular embodiment these other enzymes are well-defined (asdefined above for protease preparations).

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are 018, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

Usally fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a protease of the invention, is ananimal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (′)/0 meaning g additive per 100 g feed). Thisis so in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the inventioncomprises at least one of the below vitamins, preferably to provide anin-feed-concentration within the ranges specified in the below Table 1(for piglet diets, and broiler diets, respectively).

TABLE 1 Typical vitamin recommendations Vitamin Piglet diet Broiler dietVitamin A 10,000-15,000 IU/kg feed 8-12,500 IU/kg feed Vitamin D31800-2000 IU/kg feed 3000-5000 IU/kg feed Vitamin E 60-100 mg/kg feed150-240 mg/kg feed Vitamin K3 2-4 mg/kg feed 2-4 mg/kg feed Vitamin B12-4 mg/kg feed 2-3 mg/kg feed Vitamin B2 6-10 mg/kg feed 7-9 mg/kg feedVitamin B6 4-8 mg/kg feed 3-6 mg/kg feed Vitamin B12 0.03-0.05 mg/kgfeed 0.015-0.04 mg/kg feed Niacin 30-50 mg/kg feed 50-80 mg/kg feed(Vitamin B3) Pantothenic 20-40 mg/kg feed 10-18 mg/kg feed acid Folicacid 1-2 mg/kg feed 1-2 mg/kg feed Biotin 0.15-0.4 mg/kg feed 0.15-0.3mg/kg feed Choline 200-400 mg/kg feed 300-600 mg/kg feed chloride

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg.

WO 01/58275 corresponds to U.S. Ser. No. 09/779,334 which is herebyincorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Destillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can e.g. be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, for mashfeed a solid or liquid enzyme formulation may be added before or duringthe ingredient mixing step. For pelleted feed the (liquid or solid)protease/enzyme preparation may also be added before or during the feedingredient step. Typically a liquid protease/enzyme preparation is addedafter the pelleting step. The enzyme may also be incorporated in a feedadditive or premix.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of0.5-25 mg enzyme protein per kg animal diet. The protease should ofcourse be applied in an effective amount, i.e. in an amount adequate forimproving hydrolysis, digestibility, and/or improving nutritional valueof feed. It is at present contemplated that the enzyme is administeredin one or more of the following amounts (dosage ranges): 0.01-200;0.01-100; 0.5-100; 1-50; 5-100; 10-100; 0.05-50; or 0.10-10—all theseranges being in mg protease protein per kg feed (ppm).

For determining mg protease protein per kg feed, the protease ispurified from the feed composition, and the specific activity of thepurified protease is determined using a relevant assay (see underprotease activity, substrates, and assays). The protease activity of thefeed composition as such is also determined using the same assay, and onthe basis of these two determinations, the dosage in mg protease proteinper kg feed is calculated.

The same principles apply for determining mg protease protein in feedadditives. Of course, if a sample is available of the protease used forpreparing the feed additive or the feed, the specific activity isdetermined from this sample (no need to purify the protease from thefeed composition or the additive).

Detergent Compositions

The protease of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the protease of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas another protease, such as alkaline proteases from Bacillus, a lipase,a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alaccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g. fromH. lanuginosa (T. lanuginosus) as described in EP 258068 and EP 305216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia(EP 331376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp.strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al.(1993), Biochemica et Biophysica Acta, 1131, 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Otherexamples are lipase variants such as those described in WO 92/05249, WO94/01541, EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO 95/30744,WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.Preferred commercially available lipase enzymes include Lipolase™ andLipolase Ultra™ (Novozymes A/S). Suitable amylases (alpha- and/or beta-)include those of bacterial or fungal origin. Chemically modified orprotein engineered mutants are included. Amylases include, for example,alpha-amylases obtained from Bacillus, e.g. a special strain of B.licheniformis, described in more detail in GB 1,296,839. Examples ofuseful amylases are the variants described in WO 94/02597, WO 94/18314,WO 95/26397, WO 96/23873, WO 97/43424, WO 00/60060, and WO 01/66712,especially the variants with substitutions in one or more of thefollowing positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181,188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.Commercially available amylases are Natalase™, Supramyl™, Stainzyme™,Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes NS), Rapidase™ andPurastar™ (from Genencor International Inc.).

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495257, EP 531372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and WO99/01544. Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes NS), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g. from C. cinereus, and variants thereof as those describedin WO 93/24618, WO 95/10602, and WO 98/15257. Commercially availableperoxidases include Guardzyme™ (Novozymes).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e. a separate additive or a combined additive, canbe formulated e.g. as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

When included therein the detergent may contain a hydrotrope, which is acompound that solubilises hydrophobic compounds in aqueous solutions (oroppositely, polar substances in a non-polar environment). Typically,hydrotropes have both hydrophilic and a hydrophobic character (so-calledamphiphilic properties as known from surfactants); however the molecularstructure of hydrotropes generally do not favor spontaneousself-aggregation, see, e.g., review by Hodgdon and Kaler, 2007, CurrentOpinion in Colloid & Interface Science 12: 121-128. Hydrotropes do notdisplay a critical concentration above which self-aggregation occurs asfound for surfactants and lipids forming miceller, lamellar or otherwell defined meso-phases. Instead, many hydrotropes show acontinuous-type aggregation process where the sizes of aggregates growas concentration increases. However, many hydrotropes alter the phasebehavior, stability, and colloidal properties of systems containingsubstances of polar and non-polar character, including mixtures ofwater, oil, surfactants, and polymers. Hydrotropes are classically usedacross industries from pharma, personal care, food, to technicalapplications. Use of hydrotropes in detergent compositions allow forexample more concentrated formulations of surfactants (as in the processof compacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate(STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers,sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodiumethylhexyl sulfate, and combinations thereof.

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,or a peptide aldehyde as described in, e.g., WO 2010/055052, and thecomposition may be formulated as described in e.g. WO 92/19709 and WO92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01-100 mg of enzyme protein per liter of washliqour, preferably 0.05-5 mg of enzyme protein per liter of wash liqour,in particular 0.1-1 mg of enzyme protein per liter of wash liqour.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202.

Nucleic Acid Constructs, Expression Vectors, Recombinant Host Cells, andMethods for Production of Proteases

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides encoding the proteases of the invention.

The present invention also relates to methods of producing a protease,comprising: (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the proten.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides and fused polypeptides.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.For example, the protein may be an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Assays: Protease Assays:

1) Suc-AAPF-pNA assay:

-   -   pNA substrate: Suc-AAPF-pNA (Bachem L-1400).

Temperature: Room temperature (25° C.)

-   -   Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES,        100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted        to pH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and        11.0 with HCl or NaOH.

20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μlassay buffer. The assay was started by adding 100 μl pNA substrate (50mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% TritonX-100). The increase in OD₄₀₅ was monitored as a measure of the proteaseactivity.

2) Protazyme AK Assay:

-   -   Substrate: Protazyme AK tablet (cross-linked and dyed casein;        from Megazyme)    -   Temperature: controlled (assay temperature).    -   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES,        100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 6.5        or pH 7.0.

A Protazyme AK tablet was suspended in 2.0 ml 0.01% Triton X-100 bygentle stirring. 500 μl of this suspension and 500 μl assay buffer weredispensed in an Eppendorf tube and placed on ice. 20 μl protease sample(diluted in 0.01% Triton X-100) was added. The assay was initiated bytransferring the Eppendorf tube to an Eppendorf thermomixer, which wasset to the assay temperature. The tube was incubated for 15 minutes onthe Eppendorf thermomixer at its highest shaking rate (1400 rpm.). Theincubation was stopped by transferring the tube back to the ice bath.Then the tube was centrifuged in an ice cold centrifuge for a fewminutes and 200 μl supernatant was transferred to a microtiter plate.OD₆₅₀ was read as a measure of protease activity. A buffer blind wasincluded in the assay (instead of enzyme).

3) Suc-AAPX-pNA Assay:

-   -   pNA substrates: Suc-AAPA-pNA (Bachem L-1775)        -   Suc-AAPR-pNA (Bachem L-1720)        -   Suc-AAPD-pNA (Bachem L-1835)        -   Suc-AAPI-pNA (Bachem L-1790)        -   Suc-AAPM-pNA (Bachem L-1395)        -   Suc-AAPV-pNA (Bachem L-1770)        -   Suc-AAPL-pNA (Bachem L-1390)        -   Suc-AAPE-pNA (Bachem L-1710)        -   Suc-AAPK-pNA (Bachem L-1725)        -   Suc-AAPF-pNA (Bachem L-1400)    -   Temperature: Room temperature (25° C.)    -   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES,        100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 9.0.

20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μlassay buffer. The assay was started by adding 100 μl pNA substrate (50mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% TritonX-100). The increase in OD₄₀₅ was monitored as a measure of the proteaseactivity.

Soybean-Maize Meal Assay (SMM Assay)

An end-point assay using soybean-maize meal as substrate was used forobtaining the activity profile of the proteases at pH 3-7.

Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCAPS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted using HCl orNaOH to pH-values 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 whenmixing 10 ml assay buffer with 1 g soybean-maize meal (30:70 ratio).

2 mL soybean-maize meal slurry is mixed for 30 min before proteaseaddition and incubation for 3 hours at 40° C. (500 rpm). Protease isadded via 100 μl 100 mM sodium acetate (NaAc) buffer (9.565 g/l NaAc,1.75 g/l acetic acid, 5 mM CaCl₂, 0.01% BSA, 0.01% Tween20, pH 6.0).Supernatant are collected after centrifugation (10 min, 4000 rpm, 0° C.)and protein activity is determined using a colorimetric assay based onthe o-phthat-dialdehyde (OPA) method essentially according to Nielsen etal. (Nielsen, P M, Petersen, D, Dampmann, C. Improved method fordetermining food protein degree of hydrolysis. J Food Sci, 2001, 66:642-646). This assay detects free α-amino groups and hence proteaseactivity can be measured as an increase in absorbance. First 500 μl ofeach supernatant is filtered through a 100 kDa Microcon filter bycentrifugation (60 min, 11,000 rpm, 5° C.). The samples are diluted 10×in deionized water and 25 μl of each sample is loaded into a 96 wellmicrotiter plate (5 replicates). Finally 200 μl OPA reagent is dispensedinto all wells and the plate is shaken (10 sec, 750 rpm) and absorbancemeasured at 340 nm. The level of protease activity is calculated as thedifference between absorbance in the enzyme treated sample and the blanksample.

Results are provided in Example 4 below

In Vitro Digestion Assay

An in vitro digestion assay was used to evaluate the effect of theproteases on a feed substrate (soybean-maize meal) in a setup designedto simulate digestion in monogastric animals.

The incubation process consisted of a gastric digestion phase withporcine pepsin (SP7000, Sigma-Aldrich, St. Louis, Mo., USA) at pH 3followed by a short duodenal incubation at pH 3.8 and a small intestinalincubation with pancreatin (8×USB, P-7545, Sigma-Aldrich, St. Louis,Mo., USA) at pH 7.0.

The in vitro digestion was performed using an automated system based ona Gilson liquid handler (Biolab, Denmark). For each sample 0.8 g feedwas weighed into a tube and all tubes were placed in the liquid handler(40° C., 500 rpm). Additions of solutions as well as pH measurementswere performed automatically. At time 0 min, 4.1 mL HCl (24 mM CaCl₂)was added to reach pH 3.0 in the solution. At time 30 min 0.5 ml HCl (24mM CaCl₂, 3000 U pepsin/g feed) and 100 μL of a 100 mM sodium acetatebuffer (258.6 g NaAc per litre, 0.57% acetic acid, pH 6.0) was added. Attime 90 min 900 μL NaOH was added to reach pH ˜3.8 and at time 120 min400 μL of a 1 M NaHCO₃ solution containing 6.5 mg pancreatin/g feed wasadded leading to pH 6.8 in the solution. The pH was measured at time 30,60, 90, 115, 120 and 180 min. The test proteases were added via the 100μl NaAc buffer at time 30 min.

The level of soluble crude protein (N×6.25) measured using a LECO FP-528protein/nitrogen analyzer, was used as an indication of proteaseefficacy in the assay. Statistics: Statistical analysis of theparameters registered was performed using an analysis of variance(ANOVA) procedure and comparison of means was done using the Tukey test(α=0.05) provided by the ANOVA procedure (SAS, JMP® 5 AdministratorsGuide to Annually Licensed Windows, Mackintosh, and Linux Versions,Release 5.1. SAS Institute, Cary, N.C. (2003)).

Results are provided in Example 5 below.

Strains

Kribbella solani, isolate 067P2, was isolated from a soil sample fromthe United Kingdom obtained from Warwick University in 1990.Kribbella aluminosa, isolate 05C3Y, was isolated from a sample fromChina provided to Novozymes in 2009 under contract with Yunnan Instituteof Microbiology; Kunming.

Example 1 DNA-Preparation and Sequencing of the Kribbella solani and theKribbella aluminosa Genome

Chromosomal DNA of Kribbella solani and Kribbella aluminosa was isolatedby QIAamp DNA Blood Mini Kit” (Qiagen, Hilden, Germany). 5 ug ofchromosomal DNA of each strain were sent for genome sequencing atFASTERIS SA, Switzerland. The genomes were sequenced by IlluminaSequencing. The genome sequences were analysed for secreted S1 proteasesand the two S1 proteases (SEQ ID:1/SEQ ID:2 and SEQ ID: 3/SeqID:4) whereidentified.

Expression of Kribbella solani and Kribbella aluminosa S1 Peptidases

A linear integration vector-system was used for the expression cloningof two 51 peptidase genes from Kribbella solani (SEQ ID NO: 1) andKribbella aluminosa (SEQ ID NO: 3), respectively. The linear integrationconstruct was a PCR fusion product made by fusion of the gene betweentwo Bacillus subtilis homologous chromosomal regions along with a strongpromoter and a chloramphenicol resistance marker. The fusion was made bySOE PCR (Horton et al., 1989, Engineering hybrid genes without the useof restriction enzymes, gene splicing by overlap extension, Gene 77:61-68). The SOE PCR method is also described in patent application WO2003/095658. The gene was expressed under the control of a triplepromoter system (as described in WO 99/43835), consisting of thepromoters from Bacillus licheniformis alpha-amylase gene (amyL),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillusthuringiensis crIIIA promoter including stabilizing sequence. The genecoding for chloramphenicol acetyl-transferase was used as marker(described in, e.g., Diderichsen et al., 1993, A useful cloning vectorfor Bacillus subtilis, Plasmid 30: 312). The final gene constructs wereintegrated on the Bacillus chromosome by homologous recombination intothe pectate lyase locus.

The gene fragments of the two genes were amplified from chromosomal DNAof the two strains with specific primers (KS-forward (SEQ ID NO: 9) andKS-reverse (SEQ ID NO: 10) for the S1 protease from Kribbella solani andKA-forward (SEQ ID NO: 11) and KA-reverse (SEQ ID NO: 12) for the S1protease from Kribbella aluminosa. The upstream flanking fragment wasamplified with the primers 260558 (SEQ ID NO: 13) and iMB1361Uni2 (SEQID NO: 14) and the downstream flanking fragment was amplified with theprimers 260559 (SEQ ID NO: 15) and oth435 (SEQ ID NO: 16) from genomicDNA of the strain iMB1361 (described in patent application WO2003/095658).

Both S1 peptidase were expressed with a Bacillus lentus secretion signal(with the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA(SEQ ID NO: 17) replacing the native secretion signals. The signal wasplaced on the upstream flanking fragment. The forward primers weredesigned so that the genes were amplified from the signal peptidecleavage site and they had 26 bp overhangs and the reverse primerscontained an overhang consisting of 24-27 bp (the overhangs are shown initalic in the table below). These overhangs were each complementary topart of one or the other of the two linear vector fragments and was usedwhen the gene fragments and the vector fragments were assembled(described below). All primers used are listed in Table 2 below.

The gene fragments were amplified using a proofreading polymerasePHUSION™ DNA Polymerase (Finnzymes, Finland) according to themanufacturer's instructions. The two flanking DNA fragments wereamplified with “Expand High Fidelity PCR System” (Roche-Applied-Science)according to standard procedures (following the manufacturer'srecommendations). The PCR conditions were as follows for Kribbellaaluminosa S1 gene: 98° C. for 30 sec. followed by 35 cycles of (98° C.for 10 sec, 54° C. for 20 sec, 72° C. for 1.5 min) and ending with onecycle at 72° C. for 10 min. The PCR conditions were as follows for theKribbella solani S1 gene: 98° C. for 30 sec. followed by 35 cycles of(98° C. for 10 sec, 72° C. for 20 sec, 72° C. for 45 sec.) and endingwith one cycle at 72° C. for 10 min. For both expression constructs the3 PCR fragments were subjected to a subsequent Splicing by OverlapExtension (SOE) PCR reaction to assemble the 3 fragments into one linearvector construct.

This was done by mixing the 3 fragments in equal molar ratios and a newPCR reaction were run under the following conditions: initial 2 min. at94° C., followed by 10 cycles of (94° C. for 15 sec., 55° C. for 45sec., 68° C. for 5 min.), 10 cycles of (94° C. for 15 sec., 55° C. for45 sec., 68° C. for 8 min.), 15 cycles of (94° C. for 15 sec., 55° C.for 45 sec., 68° C. for 8 min. in addition 20 sec. extra pr cycle).After the 1^(st) cycle the two end primers 260558 and 260559 were added(20 pMol of each). Two μl of each of the PCR products were transformedinto Bacillus subtilis. Transformants were selected on LB platessupplemented with 6 μg of chloramphenicol per ml. Two recombinantBacillus subtilis clones each containing one of the integratedexpression constructs were grown in liquid cultures. The enzymecontaining supernatants were harvested and the two enzymes purified asdescribed in Example 2.

TABLE 2 Primers used Amplification SPECIFIC SPECIFIC of PRIMER FORWARDPRIMER REVERSE Kribbella  KS FORWARD  KS REVERSE  solani (SEQ ID NO: 9)(SEQ ID NO: 10) S1 peptidase 5′ 5′ CTTTTAGTTCATCGATCGCATCGGGGGCCAAGGCCGGTTTTTTATGT CT GCACCGGTGAACCCGTCCGCG TTTAGACGCTGACGCCGTAGCG3′ GGAGAG 3′ Kribbella KA forward (SEQ ID NO: 11)KA reverse (SEQ ID NO: 12) aluminosa S1 5′CTTTTAGTTCATCGATCGCATCG 5′CCAAGGCCGGTTTTTTATGTTTC peptidase GCT GCACCGGTCGACCCGTCC 3′A GTAGACGCTCACGCCGT 3′ Upstream 260558: (SEQ ID NO: 13)iMB1361Uni2 (SEQ ID NO: 14) flanking 5′ GAGTATCGCCAGTAAGGGGCG 5′AGCCGATGCGATCGATGAACTA fragment 3′ 3′ Downstream OTH435 (SEQ ID NO: 15)260559: (SEQ ID NO: 16) flanking 5′ 5′ fragment TAAAACATAAAAAACCGGCCTTGGGCAGCCCTAAAATCGCATAAAGC C3′ 3′

Example 2 Purification of the Proteases

Purification of the S1A Protease from Kribbella solani

The culture broth was centrifuged (20000×g, 20 min) and the supernatantwas carefully decanted from the precipitate. The supernatant wasfiltered through a Nalgene 0.2 μm filtration unit in order to remove therest of the Bacillus host cells. The 0.2 μm filtrate was transferred to50 mM H₃BO₃, 20 mM CH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5 on a G25 Sephadexcolumn (from GE Healthcare). The G25 sephadex transferred enzyme wasslightly turbid and was filtered through a GF/A glass microfiber filter(from Whatman). The clear filtrate was applied to a SP-sepharose FFcolumn (from GE Healthcare) equilibrated in 50 mM H₃BO₃, 20 mMCH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5. After washing the column extensivelywith the equilibration buffer, the protease was eluted with a linearNaCl gradient (0-->0.5 M) in the same buffer over five column volumes.Fractions from the column were analysed for protease activity (using theSuc-AAPF-pNA assay at pH 9). The protease peak was pooled and solidammonium sulphate was added to the pool to a final ammonium sulphateconcentration of 1.8M (NH₄)₂SO₄. The ammonium sulphate adjusted pool wasapplied to a Phenyl-sepharose FF (high sub) column (from GE Healthcare)equilibrated in 100 mM H₃BO₃, 10 mM MES/NaOH, 2 mM CaCl₂, 1.8M(NH₄)₂SO₄, pH 6.0. After washing the column extensively with theequilibration buffer, the protease was eluted with a linear gradientover eight column volumes between the equilibration buffer and 100 mMH₃BO₃, 10 mM MES/NaOH, 2 mM CaCl₂, pH 6.0 with 25% (v/v) 2-propanol.Fractions from the column were analysed for protease activity (using theSuc-AAPF-pNA assay at pH 9). The protease peak was pooled and the poolwas transferred to 50 mM H₃BO₃, 20 mM CH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5on a G25 Sephadex column (from GE Healthcare). The G25 sephadextransferred enzyme was applied to a SOURCE S column (from GE Healthcare)equilibrated in 50 mM H₃BO₃, 20 mM CH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5.After washing the column extensively with the equilibration buffer, theprotease was eluted with a linear NaCl gradient (0-->0.5 M) in the samebuffer over twenty column volumes. Fractions from the column wereanalysed for protease activity (using the Suc-AAPF-pNA assay at pH 9)and active fractions were further analysed by SDS-PAGE. Fractions, whereonly one band was seen on the coomassie stained SDS-PAGE gel, werepooled and transferred to 100 mM H₃BO₃, 10 mM MES/NaOH, 2 mM CaCl2, pH6.0 on a G25 Sephadex column (from GE Healthcare). The G25 sephadextransferred enzyme was the purified preparation and was used for furthercharacterization.

Purification of the S1A Protease from Kribbella aluminosa

The culture broth was centrifuged (20000×g, 20 min) and the supernatantwas carefully decanted from the precipitate. The supernatant wasfiltered through a Nalgene 0.2 μm filtration unit in order to remove therest of the Bacillus host cells. The 0.2 μm filtrate was transferred to10 mM succinic acid/NaOH, 1 mM CaCl₂, pH 5.0 on a G25 Sephadex column(from GE Healthcare). The G25 sephadex transferred enzyme was slightlyturbid and was filtered through a GF/A glass microfiber filter (fromWhatman). The clear filtrate was applied to a SP-sepharose FF column(from GE Healthcare) equilibrated in 10 mM succinic acid/NaOH, 1 mMCaCl₂, pH 5.0. After washing the column extensively with theequilibration buffer, the protease was eluted with a linear NaClgradient (0-->0.5 M) in the same buffer over five column volumes.Fractions from the column were analysed for protease activity (using theSuc-AAPF-pNA assay at pH 9). The protease peak was pooled and solidammonium sulphate was added to the pool to a final ammonium sulphateconcentration of 1.2 M (NH₄)₂SO₄. The ammonium sulphate adjusted poolwas applied to a Phenyl-Toyopearl column (from TosoHaas) equilibrated in100 mM H₃BO₃, 10 mM MES/NaOH, 2 mM CaCl₂, 1.2 M (NH₄)₂SO₄, pH 6.0. Afterwashing the column extensively with the equilibration buffer, theprotease was eluted with a linear (NH₄)₂SO₄ gradient (1.2-->0 M) in thesame buffer over five column volumes. Fractions from the column wereanalysed for protease activity (using the Suc-AAPF-pNA assay at pH 9)and active fractions were further analysed by SDS-PAGE. Fractions, whereonly one band was seen on the coomassie stained SDS-PAGE gel, werepooled and the pool was transferred to 10 mM succinic acid/NaOH, 1 mMCaCl₂, pH 5.0 on a G25 Sephadex column (from GE Healthcare). The G25sephadex transferred enzyme was applied to a SOURCE S column (from GEHealthcare) equilibrated in 10 mM succinic acid/NaOH, 1 mM CaCl₂, pH5.0. After washing the column extensively with the equilibration buffer,the protease was step eluted with 10 mM succinic acid/NaOH, 1 mM CaCl₂,0.5 M NaCl, pH 5.0. The eluted peak from the column was the purifiedpreparation and was used for further characterization.

Example 3 Characterization of the S1A Proteases from Kribbella

The Suc-AAPF-pNA assay was used for obtaining the pH-activity profileand the pH-stability profile (residual activity after 2 hours atindicated pH-values). For the pH-stability profile the protease wasdiluted 10× in the different Assay buffers to reach the pH-values ofthese buffers and then incubated for 2 hours at 37° C. After incubation,the pH of the protease incubations was transferred to the same pH-value,before assay for residual activity, by dilution in the pH 9.0 Assaybuffer. The Protazyme AK assay was used for obtaining thetemperature-activity profile at pH 6.5 (Kribbella solani) or at pH 7.0(Kribbella aluminosa). The Suc-AAPX-pNA assay and ten differentSuc-AAPX-pNA substrates were used for obtaining the P1-specificity ofthe enzymes at pH 9.0.

The results are shown in Tables 3-6 below. For Table 3, the activitiesare relative to the optimal pH for the enzymes. For Table 4, theactivities are residual activities relative to a sample, which was keptat stable conditions (5° C., pH 9.0). For Table 5, the activities arerelative to the optimal temperature at pH 6.5 or pH 7.0 for the enzymes.For Table 6, the activities are relative to the best substrate(Suc-AAPF-pNA) for the enzymes.

TABLE 3 pH-activity profile Kribbella solani Kribbella aluminosa pH S1Aprotease S1A protease Protease 10R 2 0.00 0.00 3 0.01 0.01 0.00 4 0.040.05 0.02 5 0.15 0.19 0.07 6 0.48 0.49 0.21 7 0.74 0.72 0.44 8 0.92 0.930.67 9 0.98 1.00 0.88 10 1.00 0.97 1.00 11 0.91 0.90 0.93

TABLE 4 pH-stability profile (residual activity after 2 hours at 37° C.)Kribbella solani Kribbella aluminosa pH S1A protease S1A proteaseProtease 10R 2 0.94 0.82 0.78 3 1.00 1.04 1.03 4 0.99 1.00 0.99 5 1.001.06 1.00 6 1.02 0.98 1.03 7 0.99 1.00 1.01 8 0.99 0.97 0.98 9 0.91 0.980.99 10 0.38 0.97 0.99 11 0.00 0.92 0.86 After 2 hours 1.00 1.00 1.00 at5° C. (at pH 9) (at pH 9) (at pH 9)

TABLE 5 Temperature activity profile at pH 6.5 or pH 7 Temp Kribbellasolani S1A Kribbella aluminosa Protease 10R (° C.) protease (pH 6.5) S1Aprotease (pH 7) (pH 6.5) 15 0.00 0.01 0.01 25 0.02 0.01 0.02 37 0.040.02 0.06 50 0.14 0.11 0.13 60 0.39 0.36 0.35 70 1.00 1.00 0.96 80 0.400.98 1.00 90 — 0.20 0.18

TABLE 6 P1-specificity on 10 Suc-AAPX-pNA substrates at pH 9 Kribbellasolani Kribbella aluminosa Suc-AAPX-pNA S1A protease S1A proteaseProtease 10R Suc-AAPA-pNA 0.13 0.15 0.13 Suc-AAPR-pNA 0.15 0.17 0.09Suc-AAPD-pNA 0.01 0.00 0.00 Suc-AAPI-pNA 0.00 0.00 0.00 Suc-AAPM-pNA0.35 0.37 0.78 Suc-AAPV-pNA 0.01 0.01 0.01 Suc-AAPL-pNA 0.21 0.19 0.18Suc-AAPE-pNA 0.00 0.00 0.00 Suc-AAPK-pNA 0.08 0.09 0.08 Suc-AAPF-pNA1.00 1.00 1.00Other Characteristics for the S1A Protease from Kribbella solani

Inhibitor: PMSF.

The relative molecular weight as determined by SDS-PAGE was approx.M_(r)=23 k Da.

The molecular weight determined by Intact molecular weight analysis was18900.5 Da.

The mature sequence (from MS-EDMAN data and P23BSS sequence) was asindicated in SEQ ID NO: 2:

The calculated molecular weight from this mature sequence was 18900.5Da.

Other Characteristics for the S1A Protease from Kribbella aluminosa

Inhibitor: PMSF.

The relative molecular weight as determined by SDS-PAGE was approx.M_(r)=21 kDa.

The molecular weight determined by Intact molecular weight analysis was19078.1 Da.

The mature sequence (from MS-EDMAN data and P23XDA sequence) was asindicated in SEQ ID NO: 4

The calculated molecular weight from this mature sequence was 19077.7Da.

Example 4 Protease Activity in Soybean-Maize Meal Assay (SMM Assay)

A soybean-maize meal assay was used to describe the activity of theproteases on a substrate relevant for animal feed. The results are shownin Table 7 below. The maximum activity for each protease is set to 1.00and the other values are represented as relative to the maximumactivity. The proteases of the invention show a lower pH optimum onsoybean-maize meal than 10R and a higher relative activity in the broadphysiological pH range from 3-7. This indicates a possibility for theproteases of the invention to hydrolyse diet protein in the entiredigestive tract of pigs and poultry. The pH in the gastrointestinaltract varies from acidic (typically pH 2-4) in the stomach of pigs andproventriculus and gizzard of poultry to pH 4-6 in the crop of poultryand pH 6-7 in the small intestine of pigs and poultry.

TABLE 7 Relative protease activity on soybean-maize meal at pH 3.0, 4.0,5.0, 6.0 and 7.0 Kribbella solani Kribbella aluminosa pH S1A proteaseS1A protease Protease 10R 3.0 0.55 0.41 0.08 4.0 0.47 0.53 0.10 5.0 0.820.83 0.24 6.0 1.00 1.00 0.62 7.0 0.76 0.83 1.00

Example 5 In Vitro Digestion Assay

A simulated gastro-intestinal digestion assay was performed to evaluatethe potential of proteases for increasing protein digestibility inmonogastric animals. The effect of the proteases was measured as anincrease in protein solubilization. The results are shown in Table 8below. The S1A protease from K. solani increased the amount of solubleprotein in the samples indicating protein hydrolysis, however not to thesame level as for protease 10R. A logical explanation for this is thatthe in vitro digestion incubation as designed for this study includes 4hours incubation at pH 7 and only 1½ hour incubation at pH≦6, the pHarea where the K. solani S1A protease of the invention has an advantageabove that of protease 10R.

TABLE 8 The level of soluble protein as percent of total protein in invitro digestion samples after treatment with Kribbella solani S1Aprotease or protease 10R Soluble protein of total (%) Enzyme (mg enzymeprotein/kg feed) Average¹ Standard deviation No enzyme 93.45^(b) 2.06Kribbella solani S1A protease (100) 97.64^(a) 1.06 Protease 10R (100)100.64^(a) 1.75 ¹Different superscript letters indicate significantdifferences (P < 0.05).

Example 6 Thermostability

An aliquot of the protein sample of protease (purified as described inExample 2) is either desalted or buffer-changed into 20 mM Na-acetate,pH 4.0 using a prepacked PD-10 column or dialysed against 2×500 ml 20 mMNa-acetate, pH 4.0 at 4° C. in a 2-3 h step followed by an overnightstep. The sample is 0.45 μm filtered and diluted with buffer to approx.2 A280 units. The dialysis buffer is used as reference in DifferentialScanning calorimetry (DSC). The samples are degassed using vacuumsuction and stirring for approx. 10 minutes.

A DSC scan is performed on a MicroCal VP-DSC at a constant scan rate of1.5° C./min from 20-90° C. Data-handling is performed using the MicroCalOrigin software (version 4.10), and the denaturation temperature, T_(d)(also called the melting temperature, T_(m)) is defined as thetemperature at the apex of the peak in the thermogram.

Example 7 Steam Stability

Residual activity of the protease after steam treatment may be evaluatedusing the following assay.

In these experiments a modified set-up is used whereby the steam isprovided from a steam generator and led into the box. The samples placedon a plate are inserted into the box through a drawer when thetemperature has reached ca. 93-94° C. Upon the insertion of the samplesthe temperature drops 4° C. Incubation is performed for 30 seconds whilethe temperature remains approximately constant at 90° C. Thereafter theplate is quickly removed from the box, the samples placed on ice,re-suspended and evaluated with respect to protease activity using e.g.the Suc-AAPF-pNA or o-Phthaldialdehyde (OPA) assay. Each enzyme sampleis compared to a similar sample that had not been steam treated in orderto calculate residual activity.

Example 8 Pelleting Stability Tests

The enzyme granulation is performed in a manner as described in U.S.Pat. No. 4,106,991, Example 1. The obtained granulate is dried in afluid bed to a water content below 1% and sifted to obtain a productwith the particle range 250 μm to 850 μm. Finally, the product is coatedwith palm oil and calcium carbonate in a manner as described in U.S.Pat. No. 4,106,991, Example 22.

Approximately 50 g enzyme granulate is pre-mixed with 10 kg feed for 10minutes in a small horizontal mixer. This premix is mixed with 90 kgfeed for 10 minutes in a larger horizontal mixer. From the mixer thefeed is led to the conditioner (a cascade mixer with steam injection) ata rate of approximately 300 kg/hour. The conditioner heats up the feedto 95° C. (measured at the outlet) by injecting steam. The residencetime in the conditioner is 30 seconds. From the conditioner the feed isled to a Simon Heesen press equipped with 3.0×35 mm horizontal die andpressed to pellets with a length of around 15 mm. After the press thepellets are placed in an air cooler and cooled for 15 minutes.

The protease activity is measured using the Suc-AAPF-pNA assay prior topelleting and in the feed pellets after pelleting. Pelleting stabilityis determined by comparing the protease activity in pelleted feedrelative to the activity in non-pelleted feed.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated polypeptide having protease activity, selected from thegroup consisting of: (a) a polypeptide having at least 85% sequenceidentity to the mature polypeptide of SEQ ID NO: 2 and/or SEQ ID NO: 4;(b) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, and/or (ii) themature polypeptide coding sequence of SEQ ID NO: 3, or (iii) thefull-length complementary strand of (i) or (ii); (c) a polypeptideencoded by a polynucleotide having at least 86% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3;and/or (d) a variant comprising a substitution, deletion, and/orinsertion of one or more (several) amino acids of the mature polypeptideof SEQ ID NO: 2; and/or SEQ ID NO:
 4. 2-7. (canceled)
 8. An isolatedpolynucleotide encoding the polypeptide of claim
 1. 9. A nucleic acidconstruct or expression vector comprising the polynucleotide of claim 8operably linked to one or more (several) control sequences that directthe production of the polypeptide in an expression host cell.
 10. Arecombinant expression host cell comprising a polynucleotide of claim 8operably linked to one or more control sequences that direct theproduction of the polypeptide.
 11. A method of producing the polypeptideof claim 1, comprising: (a) cultivating a cell, which in its wild-typeform produces the polypeptide, under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.
 12. A method ofproducing the polypeptide having protease activity, comprising: (a)cultivating a host cell of claim 10 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.13-15. (canceled)
 16. A method for improving the nutritional value of ananimal feed, wherein at least one polypeptide of claim 1 is added to thefeed.
 17. An animal feed additive comprising (a) at least onepolypeptide of claim 1; and (b) at least one fat-soluble vitamin, and/or(c) at least one water-soluble vitamin, and/or (d) at least one tracemineral.
 18. The animal feed additive of claim 17, which furthercomprises one or more amylases, phytases, xylanases, galactanases,alpha-galactosidases, proteases, phospholipases; beta-glucanases, or anymixture thereof.
 19. An animal feed having a crude protein content of 50to 800 g/kg and comprising at least one polypeptide of claim
 1. 20. Amethod for the treatment of proteins, comprising the step of adding atleast one polypeptide of claim 1 to at least one protein or proteinsource.
 21. The method of claim 20, wherein soybean is included amongstthe at least one protein source.
 22. (canceled)
 23. A detergentcomposition comprising at least one polypeptide of claim
 1. 24. Thedetergent composition of claim 23, wherein the composition comprises oneor more further enzymes.
 25. The detergent composition of claim 24,wherein the further enzymes are selected from the group comprisingproteases, amylases, lipases, cutinases, cellulases, endoglucanases,xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes,haloperoxygenases, catalases and mannanases, or any mixture thereof.